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RAPD (random amplified polymorphic DNA) primers for differentiating pear varieties and application of primers

A variety and primer pair technology, applied in the field of molecular biology, can solve the problems of large operation volume, lack of practicability, and inability to visually display the primers of multiple varieties, and achieve the effect of simple operation and wide versatility.

Active Publication Date: 2015-05-27
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the existing research work on species differentiation and germplasm identification using RAPD technology, computer software is often used to draw digital fingerprints, and cluster trees are obtained through cluster analysis. The primers used for each variety, and the identification process cannot be displayed, which requires a large amount of operation and lacks practicability

Method used

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  • RAPD (random amplified polymorphic DNA) primers for differentiating pear varieties and application of primers
  • RAPD (random amplified polymorphic DNA) primers for differentiating pear varieties and application of primers
  • RAPD (random amplified polymorphic DNA) primers for differentiating pear varieties and application of primers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Extraction of DNA.

[0043] The pear variety samples of the present invention are all important pear varieties that are common in the market. These 22 varieties include: Cuiguan, Cuilu, Zhongli No. 1, Cuiyu, Chuxialv, Zaoguan, Zaojinsu, Jinjing, Yulu, Huangguan , Yuluxiang, Jiyu, Hongcrispy, Hanhong, Hanxiang, Yuanhuang, Xizigreen, Sucui No. 1, Sucui No. 2, Fengshui, Qiuzili, Fengxiang.

[0044] Take about 0.2g of young pear leaves as the material, grind with liquid nitrogen, add 500μl CTAB lysate (2% CTAB, 2M NaCl 2 , 20mM EDTA, 100mM Tris-HCl (PH=8.0) and 0.2% β-mercaptoethanol), transfer to a 1.5ml centrifuge tube, take out and cool in a 65°C water bath for 0.5-1h, add an equal volume of chloroform: isoamyl alcohol ( 24:1) After shaking the mixture gently, centrifuge the emulsion at 12000r / min for 8-10min, extract the supernatant with chloroform / isoamyl alcohol (24:1) for 1-2 times, add pre-cooled isopropanol 4 ℃ refrigerator for more than 3 hours, the c...

Embodiment 2

[0045] Example 2: RAPD-PCR reaction system and conditions.

[0046] RAPD-PCR reaction system is 20μl, containing 50ng genomic DNA, 2.0mmol L -1 MgCl 2 , 0.15mmol·L -1 dNTPs, 0.44 μmol L -1 10mer primers, 0.75U Taq DNA polymerase.

[0047] The RAPD-PCR amplification program was pre-denaturation at 94°C for 2min, denaturation at 94°C for 30S, annealing for 45S, extension at 72°C for 90S, 42 cycles, extension at 72°C for 10min, and storage at 4°C. Amplify on a PCR amplification instrument, and the amplified product adopts agarose gel electrophoresis (containing 1.2% ethidium bromide), 1 × TAE electrophoresis buffer (contains in 1L: 0.242g Tris, 0.057ml glacial acetic acid, 0.2 ml 0.25mol / L EDTA pH 8.0). Add 4 μl of 6× loading buffer (containing 0.25% bromophenol blue, 30% sucrose) to the amplified DNA sample, and perform electrophoresis at a constant voltage of 120V for 0.5-0.8 hours until the amplified DNA bands are clearly separated After that, the amplified bands were ob...

Embodiment 3

[0048] Example 3: Stringent screening of specific primers.

[0049] The three primers of the present invention are obtained through strict screening of 42 random primers originally designed. Firstly, 42 random primers were designed for the genome of the existing pear varieties, and 11 bases of RAPD random primers with good polymorphism, strong amplification and high stability were selected from them, and the band patterns were screened out by gradient PCR twice. Clear and reproducible primers. In detail, the gradient PCR reaction system is 20 μl, containing 50ng genomic DNA, 2.0mmol·L -1 MgCl 2 , 0.15mmol·L -1 dNTPs, 0.44 μmol L -1 10mer primers, 0.75U Taq DNA polymerase. The reaction was pre-denatured at 94°C for 2min, denatured at 94°C for 30S, annealed for 45S, extended at 72°C for 90S, 42 cycles, and extended at 72°C for 10min. According to 2 consecutive gradient PCRs, select the primers with clear bands and repeated PCR products, and the annealing temperature of the...

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Abstract

The invention discloses RAPD (random amplified polymorphic DNA) primers for differentiating pear varieties and application of the primers and belongs to the field of molecular markers in molecular biology. The method screens three RAPD random primers through a design. The nucleotide sequences of the primers are shown in SEQ ID NO: 1-3 for amplifying DNA of pears of unknown pears. The variety with a unique specific band is differentiated by performing statistics on the band after gel electrophoresis and a tree identification graph of a differentiated variety is established. The primers disclosed by the invention improve the stability of RAPD reaction. The primers are simple to operate and efficient and accurate and are saved, and two pear varieties such as Yuluxiang and the like can be quickly differentiated in the molecular level smoothly through PCR three times, and the obtained variety identification graph is of more intuition compared with that of a clustering tree, i.e., the primers capable of differentiating any two varieties can be quickly found according to the variety identification graph. The method can be used for realizing early evaluation of pear seedlings and further has wide universality on other species.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for quickly distinguishing pear varieties by using RAPD molecular marker technology. Background technique [0002] China is one of the centers of origin of the genus Pyrus in the world, and the cultivated area and output of pear trees rank first in the world. In recent years, with the rapid development of my country's pear industry, a large number of fine varieties have been used in pear production. However, due to the long-term artificial cultivation process, the phenomenon of pear variety strains is serious. The evaluation of genetic diversity brings inconvenience. Therefore, it is very important to establish an efficient, fast and reliable identification technology system for pear varieties for early identification of seedlings, differentiation of varieties and protection of variety rights. [0003] At present, it is difficult to effectively iden...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 许园园常有宏蔺经李慧
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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